Detection and identification of acid-fast bacilli of the genus Mycobacterium by conventional procedures requires growing the organisms from patient specimens and then testing the isolates for various phenotypic characteristics. These methods may require as long as one or more months. The development of a few highly specific molecular probes for testing cultures growing acid-fast bacilli has greatly reduced the time to identification of some mycobacterial isolates. Recently, the polymerase chain reaction (PCR) and isothermal nucleic acid amplification techniques have been used in assays that offer a high degree of specificity and reasonable sensitivity for detection of individual mycobacterium species in clinical samples. At present, there are no commercially available amplification assay systems capable of detecting all members of the genus Mycobacterium while excluding cross-reactive signals from other bacteria commonly present in clinical samples. We have developed a PCR assay using primers targeting a segment of the 165 ribosomal RNA gene. The assay is capable of amplifying DNA sequences from all members of the. genus Mycobacterium while excluding cross-reactive signals from most other bacteria commonly present in clinical samples. Individual mycobacterial species are then identified using specific europium-labeled fluorescent probes. We are currently optimizing the assay conditions to increase sensitivity. We are also examining several new nucleic acid extraction methodologies, which may greatly simplify sample preparation for mycobacterial nucleic acid detection assays. A highly sensitive, genus-wide mycobacterial nucleic acid detection system coupled with a simple, reliable sample preparation technique could greatly reduce the time to detect mycobacterial infections in patients.